Nanoparticle arrays and sensors using same

ABSTRACT

A method for enhancing the dispersibility of calixarene-stabilized nanoparticles, which can be assembled into ordered, planar arrays of monoparticulate thickness, ring-like assemblies of discrete particle count, and other configurations.

[0001] This application claims the benefits of U.S. provisionalapplication Serial No. 60/312,097 filed Aug. 14, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for enhancing thedispersibility of nanoparticles, which can be applied toward theformation of 2D nanoparticle assemblies and arrays with an orderedstructure as well as sensors using same.

BACKGROUND OF THE INVENTION

[0003] The dispersion and self-assembly of nanoparticles are consideredby many to be key methodologies in materials synthesis. For example, thenext generation of materials for nanoscale electronic and other devicesmay require production and use of nanoscale particles, such as nanoscalemetallic particles, which should be resistant to environmentaldegradation and amenable to synthesis in the form of nanostructuredmaterials. For example, the spontaneous assembly of monolayer-protectednanoparticles into periodic two-dimensional (2D) arrays is of interestsince many such arrays may demonstrate novel optical and/or electronicproperties as a function of particle size, composition and interparticlespacing in the array that may of use in optical/infrared scattering,radiation shielding, or sensing.

[0004] Hydrophobic surfactants such as alkanethiols have been used todrive the nanoparticles toward self-assembly at an aqueous interface;however, 2D array formation by this technique has so far been limited tosmall (less than 10 nm) nanoparticles. Stabilized metal particles beyondthis threshold become increasingly prone to multi-layer orthree-dimensional aggregate formation, which can be attributed to therapid increase in van der Waals attraction between particles as afunction of size and interatomic pair potentials.

SUMMARY OF THE INVENTION

[0005] The present invention provides a method for enhancing thedispersibility of calixarene-stabilized nanoparticles, which can beassembled into ordered, planar arrays of monoparticulate thickness,ring-like assemblies of discrete particle count, and othertechnologically useful configurations.

[0006] In practicing an illustrative embodiment of the invention, ananoparticle is treated with calixarenes, such as resorcinarenes, morepreferably tetrathiol resorcinarenes, having a macrocyclic, concave,multi-valent headgroup with multiple hydrocarbon tails per molecule withthe hydrocarbon tails spaced several angstroms apart. Nanoparticles canbe dispersed in liquid organic solvents or at the interface betweenorganic and aqueous liquid solvents. Nanoparticles selected from metals,alloys, magnetic materials and semiconducting materials may usedpursuant to the invention. Metal nanoparticles can be extracted fromaqueous suspension and dispersed into a nonpolar organic liquid solventmedium.

[0007] In practicing an illustrative embodiment of the invention, aplanar array is formed by introducing calixarene-treated nanoparticlesin the size range of 15 to 200 nm (nanometers) onto an gas/liquid (e.g.air/water) interface in a container to form a self-assembled,two-dimensional (2D) film array with monoparticulate thickness and longrange order, which can then be deposited as such onto a suitablesubstrate by relative movement of the substrate and the interface. Theself-assembled, nanostructured 2D array is deposited having organizedlong-range order of the nanoparticles on the substrate. Optical or otherproperties of 2D arrays formed pursuant to the invention can be tuned asa function of particle size, particle composition, and interparticlespacing, the latter being especially influential in controlling opticaland electronic properties of the arrays.

[0008] The above-described nanoparticle arrays provided pursuant toanother embodiment of the invention may find use in devices and methodsinvolving optical/infrared scattering, electromagnetic shielding,chemical sensing, chemical synthesis, and biomedical applicationsincluding drug delivery and therapeutic agents. Chemical, biomolecularand other sensors are envisioned using the above-described nanoparticlearrays pursuant to the invention.

[0009] The above objects and advantages of the invention will becomeapparent from the following description of the invention.

DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1A is a diagram of a gold nanoparticle being treated withresorcinarene designated 1, 2, 3, or 4. FIGS. 1B, 1C, and 1D each is arepresentation of a transmission electron micrograph (TEM) of aself-assembled 2D nanoparticle array of encapsulated gold particles onFormvar-coated Cu grids pursuant to the invention. In FIGS. 1B, 1C, and1D, the nanoparticles had an average particle diameter of 16, 34, and 87nm, respectively. In FIGS. 1B and 1C, the arrays were transferred onto aFormvar-coated Cu grids (300 mesh) by manual Langmuir-Schaeferhorizontal deposition. In FIG. 1D, the array was transferred bymechanically controlled vertical deposition onto a Formvar-coated Cugrid mounted on a glass slide which had been ionized just prior to use.

[0011]FIG. 2 includes extinction spectra of large gold nanoparticleplanar arrays on smooth quartz substrates for nanoparticle sizes of 16,34, 42, 70, 87, 111, and 170 nm. Substrates were oriented with thearrays facing the light source to minimize interferometric fringeeffects. Inflections in the spectra at 630 and 1400 nm are artifactsfrom instrumentation. Spectral intensities have been modulated forclarity in presentation with minimal effect on the extinction maxima.

[0012]FIGS. 3A, 3B show surface-enhanced Raman scattering as a functionof array structure. In FIG. 3A, spectra were obtained using a dispersivespectrometer at a Raman excitation wavelength of 647.1 nm by a kryptonion laser. In FIG. 3B, spectra were obtained using an FT spectrometer ata Raman excitation wavelength of 1064 nm by a Nd:VO₄ laser.

[0013]FIGS. 3C, 3D show G values (signal enhancement factors) as afunction of periodic array structure and numerical aperture at a fixedexcitation wavelength (785 nm). Error bars are equal to one standarddeviation based on replicate measurements at different positions on thesample.

[0014]FIGS. 4A, 4B, 4C, and 4D each is a representation of atransmission electron micrograph (TEM) of a self-assembled 2Dnanoparticle planar array of encapsulated gold particles pursuant to theinvention. In FIGS. 4A and 4B, particle size is 42 and 70 nm,respectively. In FIGS. 4C and 4D, particle size is 111 and 170 nm,respectively. In FIGS. 4A and 4B, the arrays were transferred onto aFormvar-coated Cu grids (300 mesh) by manual Langmuir-Schaeferhorizontal deposition. In FIGS. 4C and 4D, the arrays were transferredby mechanically controlled vertical deposition onto a Formvar-coated Cugrid mounted on a glass slide which had been ionized just prior to use.

[0015]FIG. 5 is a representation of a transmission electron micrograph(TEM) of a self-assembled 2D nanoparticle array of encapsulated goldparticles pursuant to the invention where particle size is 70 nm.

[0016]FIG. 6 shows unenhanced Raman spectra of resorcinarenetetrabromide 3 as a function of film thickness where small quantities ofthe compound were placed on a quartz substrate and heated slowly on ahot plate until melted, then were pressed into a thin film using a glasscover slip. Film thicknesses were measured using a micrometer with aresolution of 0.1 micron. Spectra were obtained using a dispersive Ramanmicroscope with a 10× objective lens at an excitation wavelength andpower of 785 nm and 20 mW by a diode laser (SDL).

[0017]FIG. 7 shows SERS difference spectra of nitrobenzene adsorbed toresorcinarene-encapsulated 170 nm particle array. Spectra were obtainedusing a dispersive Raman microscope with a 20× objective lens at anexcitation wavelength and irradiation time of 30 seconds. Spectra a andb have been shifted and magnified for clarity of presentation.Difference spectra a is after 5 minute exposure to nitrobenzene vapor.Difference spectra b is after exposure to 30 mL ofnitrobenzene-saturated air. Spectra c is Raman spectra of neatnitrobenzene. Peak assignments (cm⁻¹) are as follows: 854, β (NO₂); 1005v₁₂ (arom); 1110 v₁₃ (arom); 1345 v_(s) (NO₂).

[0018]FIG. 8 is a representation of a transmission electron micrograph(TEM) of a self-assembled 2D nanoparticle array of encapsulated cobalt(Co) particles pursuant to the invention where ring-like bracelets areself-assembled.

[0019]FIG. 9 is a schematic view of a nanoparticle array as SERS-basedsensor of cell afflux activity.

[0020]FIG. 10 is a diagram of a gold nanoparticle being treated withresorcinarene 5 or 6 which is then cross-linked.

DESCRIPTION OF THE INVENTION

[0021] The robustness and dispersion control of nanoparticles in varioussolvents (liquid media) are enhanced by employing calixarenes asstabilizing surfactants. The invention provides in one embodiment aninexpensive and reliable procedure for fabricating highly ordered,monoparticulate nanoparticle arrays and assemblies with valuable opticalproperties, and nanoparticle rings with unusual magneticcharacteristics. These physical properties can be tuned as a function ofparticle size, composition, and interparticle spacing. The inventionprovides in another embodiment a method for encapsulating nanoparticlesin robust (strongly attached), nondesorptive shells, which can be usedin a variety of applications including organic synthesis, drug delivery,and chemical and biological separations.

[0022] Calixarenes are characterized by macrocyclic polyaromaticheadgroups connected to several hydrocarbon tails (tailgroups) asdescribed by V. Bohmer in “Calixarenes Macrocycles with (Almost)Unlimited Possibilities”, Angew. Chem. Int. Ed. Engl. 34, 713-745, 1995,the teachings of which are incorporated herein by reference. Calixareneis a macrocycle formed by the condensation of phenolic and heterocyclicunits and aldehydes or derivatives thereof, characterized by a concavity(headgroup) often rimmed with heteroatoms (N, O, P, S, Cl, Se, Br, andI) or functional groups containing such atoms, and multiple substituents(tailgroups) of hydrocarbon chains, aromatic rings, polyether chains, orcombinations thereof. These can also be functionalized with heteroatomsor functional groups containing such atoms. Examples include resorcinol(1,3 hydroxybenzene)-derived calixarenes (resorcinarenes and cavitands),pyridine-derived calixarenes (pyridene-arenes), and pyrrole-derivedcalixarenes (calix-pyrroles). Exemplary calixarenes compriseresorcinol-derived calixarenes (resorcinarenes) with various functionalgroups attached either to the macrocylic headgroup (e.g. R, X=H, CH₃,CH₂SH, PPh₂, CS₂, CH₂N-dialkyl, CH₂N-alkyl-CS₂) or the tailgroups (e.g.terminal olefins, amines, thiols, alcohols, carboxylates, and otherfunctional groups or derivatives thereof).

[0023] Pursuant to the invention, adsorption of calixarene molecules tothe nanoparticle surfaces renders them hydrophobic and highly repulsiveat close range. This repulsion is partly due to entropic factors; thespacing of the tails precludes dense packing and increases the overallconformational entropy of the surfactant layer, thereby raising thebarrier against steric compression. Excellent dispersing properties havebeen demonstrated using resorcinarenes, a class of calixarenes derivedfrom resorcinol, which were able to stabilize dispersions of large goldnanoparticles in hydrocarbon liquid solutions.

[0024] For purposes of illustration and not limitation, a method forfabricating monoparticulate films via the self-organization usescalixarene surfactants designed to promote the self-organization ofmetal (e.g. gold) nanoparticles up to 170 nm (nanometer) in diameterinto hexagonally close-packed (hcp) arrays at the air-water interface.The formation of well-ordered arrays or assemblies from large goldnanoparticles is used herein for demonstrating this methodology, sincenearly all materials possess Hamaker constants smaller than that of goldand therefore are not as strongly affected by attractive interparticleforces. For purposes of illustration and not limitation,resorcinarene-derived surfactants with sulfur-functionalized headgroups;i.e. tetrabenzylthiol C11 resorcinarene 1 and tetraarylthiol C11resorcinarene 2 were used extract and disperse colloidal goldnanoparticles up to nearly 100 nm from aqueous suspensions into nonpolarliquid solvent media. These resorcinarene molecules have a large,macrocyclic, concave, multi-valent headgroup with multiple hydrocarbontails per molecule with the hydrocarbon tails spaced several angstromsapart (e.g. see FIG. 1A). The synthesis of tetrathiol resorcinarene 1 aswell as tetraarylthiol resorcinarene 2 is described below underSynthesis Procedures and in the literature for closely relatedmolecules, such as in Moran et al. “Cavitands Synthetic MoleculeVessels”, J. Am. Chem. Soc. 104, 5826-5828, 1982, and Gibb et al.“Efficient coupling of amino acid derivatives to rigid organicscaffolds: model syntheses for de novo proteins”, Tetrahedron 51,8719-8732, 1995. For example, tetrabenzylthiol C11 resorcinarene 1 wassynthesized by O,O-methylenation of tetra (2-methyl) resorcinarene atelevated temperature and pressure, followed by bromination of the metylgroups and treatment with thiourea. Tetraarylthiol resorcinarene 2 wasprepared by lithium-halogen exchange of the corresponding tetrabromidefollowed by treatment with elemental sulfur.

[0025] Preparation of Monoparticulate Films of Large Gold Nanoparticles

[0026] Subpicomolar aqueous (10⁹-10¹¹ particles/mL) ofcitrate-stabilized gold nanoparticles of narrow size dispersity withrelative standard deviations (RSDs) of ≦20% purchased from BritishBiocell International were treated briefly with a mixed-bed ion-exchangeresin (Amberlite MB-3 available from Mallinckrodt Co.), then transferredto a silanized test tube and mixed vigorously with a 1 mM solutionresorcinarene 2 in THF in equal proportions (16-42 nm particles) or in a3:4 ratio (70-170 nm particles). The number of particles used wasroughly 150% the estimated amount necessary to form an hcpmonoparticulate film across the air-water interface. The mixture wasextracted several times against toluene to remove THF and excesssurfactant. The resorcinarene-coated nanoparticles were flocculated atthe solvent interface and were densified by drawing the aqueoussuspension into a silanized glass pipet, then carefully draining thesupernatant until a minimal volume was achieved. The remainingsuspension was spread across the air-water interface of a test tube andallowed to sit undisturbed for at least 30 minutes to allow for thecomplete evaporation of toluene. The resulting monoparticulate film washighly reflective and contained domains which appeared uniform to thenaked eye.

[0027] Transfer of Gold Nanoparticle Arrays

[0028] The monoparticulate films were transferred at the air-waterinterface onto Formvar-coated TEM grids either by the manual horizontaldeposition commonly known as the Langmuir-Schaefer technique, or by slow(1 mm/mm) vertical retraction (lifting) of a partially immersedsubstrate through the air-water interface using a mechanicallycontrolled deposition arm available from Kibron, Inc. connected to theupper end of the substrate. The substrates were either heat-annealedquartz slides soaked in piranha solution just prior to deposition, orFormvar-coated TEM grids mounted on glass substrates and ionized justbefore use. AFM linescan analysis of the quartz substrates indicated apeak-to-valley roughness of approximately 1 nm. Films on quartzsubstrates were inspected for regions of optical uniformity at 40×magnification prior to SERS analysis (see below).

[0029] TEM Analysis

[0030] Gold nanoparticle arrays transferred onto Formvar-coated Cu grids(300 mesh) were analyzed by TEM using a Philips EM-300 at acceleratingvoltages of 80 or 100 kV. Particle arrays were transferred either byhorizontal deposition (average particle diameters are 16, 34, 42 and 70nm, see FIGS. 4A, 4B and FIGS. 1B, 1C) or by vertical deposition(average particle diameters are 87, 111 and 170 nm, see FIGS. 4C, 4D andFIG. 1D). TEM analyses were always performed shortly after arraytransfer. A number of particles appear to be fused; however, themajority of these (>95%) are single particles with slight lateraloverlap as a result of size dispersity, whose edges can be distinguishedin the original images. The occasional truly fused particles are mostlikely due to sintering by the high-energy electron beam duringanalysis.

[0031] Average particle diameters (d) were calculated from the digitizedareas of individual nanoparticles within the array (N≧l00) using a sizeanalysis program (SigmaScan 5.0, SPSS). With the exception of the 16 nmparticles, the RSDs (relative standard deviations) were all less than10%, indicating that the particles in the arrays were self-selecting.Fast Fourier transforms (FFTs) were performed using an Adobe Photoshopplug-in (Image Processing Toolkit, Reindeer Games). FFT analysis yieldedaverage periodicities which were comparable to or less than d, primarilybecause of apparent particle overlap and other defects in latticestructure (see FIG. 5, Table A). Errors based on calculations from thevarious harmonics in a given transform (typically first through third)were between 6% and 15%; these were also too significant to allowmeaningful estimates of interparticle distances, which were 1-5% that ofd. Therefore, average interparticle spacing parameters (δ) weredetermined from the digitized distances between nearest-neighborparticles (N=25) using a standard graphics software package (AdobePhotoshop 5.0 Adobe Systems). Indeterminate errors caused by differencesin focal length or image editing were found to be insignificant comparedto the digital resolution of the images (0.22 nm/pixel) and the standarddeviations (0.14-0.31 nm). The latter measurements were restricted tospacings between non-overlapping particles completely embedded within ahcp (hexagonal close packed) lattice, and were thus able to generatedistance parameters with a greater degree of precision. Although thesevalues are not representative of the entire array, the relative trend inδ with increasing periodicity is unmistakable. The interparticle spacingvalues of Table A demonstrate a trend toward decreasing interparticlespacing with increasing periodicity.

[0032] Estimation of Surface Enhancement Factors in SERS

[0033] Empirical signal enhancement factors were determined by comparingthe peak area integrations centered at 813 cm⁻¹ from resorcinarenetetrathiol 2 adsorbed on the gold nanoparticle arrays with thecorresponding peak integrations centered at 651 cm⁻¹ from unenhancedfilms of resorcinarene tetrabromide 3. Compounds 2 and 3 have verysimilar characteristic Raman group frequencies (equivalent peak in solid1 is at 663 cm⁻¹), but resorcinarene tetrabromide 3 was used as areference because of its greater chemical and thermal stability. Astandard graphing software package (KaleidaGraph, Synergy) was used toperform data manipulations and peak integrations. Films of variablethicknesses (18-30.5 microns±0.5 micron, see FIG. 6) were measured toascertain that signal intensities increased in proportion with filmthickness, allowing us to estimate the unenhanced Raman signals in termsof molecular density per unit area. The density of resorcinarenetetrabromide 3 in a liquid (melt) state was determined to be 1.13±0.23g/ml, corresponding to an areal density of 1.48×10¹⁸ molecules/cm² in a30.5 micron film. Obtaining areal densities of resorcinarene tetrathiol2 adsorbed to the nanoparticle arrays required some simplifyingassumptions: (i) the nanoparticle arrays all have approximately equalsurface roughness (1.8 for an ideally planar hcp array); (ii) the SERSsignals are spatially averaged and can be quantified as a function ofareal density of the adsorbate; (iii) a high percentage of thenanostructured surfaces are passivated by resorcinarene tetrathiol 1with an average surface density of 2 nm²/molecule, corresponding toapproximately 75% particle coverage and an areal density of 6×10¹³molecules/cm². The signal enhancement factor G can then be quantifiedas:$G = {{\frac{S^{array}}{S^{{thin}\quad {film}}}( \frac{D^{{thin}\quad {film}}}{D^{array}} )} = {2.46 \times {10^{4} \cdot \frac{S^{array}}{S^{{thin}\quad {film}}}}}}$

[0034] where S and D are the integrated signal and molecular density perunit area of the resorcinarenes in the unenhanced (thin film) andsurface-enhanced (array) samples. It is important to note that theenhanced Raman scattering cross sections may vary greatly betweenmolecules depending on their site of adsorption (see main text);therefore, signal enhancements determined by this method are relative atbest.

[0035] Multiple Raman spectra (N=9-11) of each nanoparticle array wereacquired at 785 nm excitation, N.A.=0.75 at different locations on thesample to ascertain signal reproducibility. The peak integration RSDsranged from 14 to 35% (see Tables B, C). The signal enhancements appearto be reproducible between replicate arrays, with occasional sampleshaving anomalously strong signal. For example, three 170-nm particlearrays transferred onto glass substrates had average G values of1.27×107, 1.28×10⁷, and 3.18×107. Signal enhancements from other arrays(d=70 nm) were also reproducible within one standard deviation of themean; however, many of the replicate samples contained high levels ofbackground fluorescence with substantial baseline distortion, preventinga more comprehensive evaluation.

[0036] SERS as Function of Wavelength and Collection Angle

[0037] Raman spectra at an excitation wavelength of 647.1 nm wererecorded using a dispersive spectrometer (SpectraCode, N.A.=0.4,sampling diameter approx. 120 micron, integration time=10 sec) and akrypton ion laser (Spectra-Physics, 50 mW at the sample). Spectra at anexcitation wavelength of 1064 nm were obtained using a FT spectrometerNicolet, N.A.=0.5, sampling diameter>1000 micron, 128 scans) and aNd:VO₄laser (500 mW). Spectra at an excitation wavelength of 785 nm wereacquired using a home-built dispersive Raman imaging microscope and adiode laser (SDL, 315 mW). MicroRaman spectra were obtained using l0×,20×, and 40× objective lenses (Olympus, N.A.=0.25, 0.40, and 0.75respectively). The laser input power, which was limited by theacceptance apertures on the back side of the objectives (overfilledcase), was measured to be 20 mW, 12 mW, and 10 mW at the sample,respectively.

[0038] Relative changes in Raman scattering cross sections as a functionof aperture were determined according to the equation:

S=(P _(D) βDK) (A _(D)Ω_(D) TQ)t _(s)

[0039] where P_(D) is the laser power at the sample, β is the Ramanscattering cross section of the analyte, D is the molecular density, andK is a geometric factor defining depth of field; A_(D) is the samplingarea, Ω_(D)=π(N.A.)² is the solid angle of collection in steradians, Tis the transmission efficiency factor, Q is the quantum efficiency, andt_(s) is the time of measurement. A_(D), Ω_(D) and K (for a thicktransparent sample such as neat benzene, change with the N.A. of theobjective lens (see Table D). Assuming all other parameters to beconstant, the changes in S/β for the 20× and 40× objectives relative tothe l0× objective are approximately 0.24 and 0.067, respectively. Therelative signal intensities from neat benzene at 992 cm¹ for the 20× and40× objectives were measured to be 0.362 and 0.038, yielding relative βvalues of 1.5 and 0.57, respectively. It should be mentioned that thesevalues are affected by the transmission efficiency of the objective lensitself; however, changes in signal which are not related to N.A. can bedescribed as a single variable for the purposes of this study.

[0040] Similar comparisons were made using a thin film of resorcinarenetetrabromide 3 and the nanoparticle arrays in which K is constant withrespect to N.A., so that changes in β are only due to P_(D), A_(D), andΩ_(D). The relative change in β for the resorcinarene thin film withtile 20× lens was determined to be almost identical to that of benzene,but it was not possible to obtain a thin film Raman signal at 40× ofsufficient quality for comparison. Therefore, the relative β from thebenzene measurement at 40× was used to estimate a value for S of thethin film at N.A.=0.75 relative to the signal obtained at N.A.=O.40:$S_{40X}^{{thin}\quad {film}} = {{{S_{20X}^{{thin}\quad {film}}( \frac{\beta_{40X}^{benzene}}{\beta_{20X}^{{thin}\quad {film}}} )}( \frac{P_{40X}}{P_{20X}} )( \frac{A_{40X}}{A_{20X}} )( \frac{\Omega_{40X}}{\Omega_{20X}} )} = {0.273 \cdot S_{20X}^{{thin}\quad {film}}}}$

[0041] Signal enhancement factors at N.A.=O.75 were then calculated andfound to be nearly an order of magnitude greater than those obtained atN.A.=0.25 (see FIG. 3D). The average relative SERS cross sections(β^(SERS) at) 20× and 40× were determined to be 3.2 and 5.0 (see TableE); the corresponding ratio of β^(SERS) to β^(thin film)(the relativechange in G as a function of aperture) is 2.0 and 8.7, respectively.TABLE A TEM analysis of hcp gold nanoparticle arrays: particle size,interparticle spacing, and FFT analysis average interparticle particlespacing Average diameter, d parameter 1st harmonic 2nd harmonic 3rdharmonic periodicity (nm) S.D. (nm) (nm) S.D. (nm) analysis (nm)analysis (nm) analysis (nm) (nm) S.D. (nm) 15.9 3.0 1.00 0.31 16.1 18.517.3 1.7 34.5 2.4 0.89 0.26 32.0 36.5 38.4 35.6 3.3 41.6 3.4 0.78 0.1640.4 43.8 41.3 3.0 70.0 5.0 0.64 0.20 69.7 73.1 71.9 71.6 1.7 87.4 6.70.57 0.22 77.7 84.7 87.4 82.1 6.2 111.5 7.9 0.40 0.14 93.2 116.5 107.6105.8 11.8 170.2 12.1 n/a n/a 153.4 170.5 143.9 155.9 13.5

[0042] TABLE B Summary of replicate measurements of Raman peakintegration at 831 cm−1 (785 nm excitation, N.A. = 0.75) Minimum MaximumParticle integrated integrated Std diameter area area samples (N ) MeanDeviation 16 nm 4326.6 6462.8 9 5329.9 872.8 34 nm 58036 90838 9 738919971 42 nm 35282 87440 9 56708 17180 70 nm 39640 102230 9 77462 23867 87nm 91328 193410 11 127950 35634 111 nm  50621 146510 9 84665 29377 170nm  176910 419880 10 283320 74676

[0043] TABLE C Replicate measurements of Raman peak integration at 831cm−1 (785 nm excitation, N.A. = 0.75) Sam- ple no. 16 nm 34 nm 42 nm 70nm 87 nm 111 nm 170 nm 1 4481 68903 68052 39640 107110 53718 309565 24700 67951 87440 100489 101649 50621 338441 3 5529 71845 35282 98144123430 77458 287732 4 5496 72133 41365 73602 91328 73037 337689 5 646186001 44676 70952 108210 79689 236445 6 4327 79207 48807 44696 94453109414 211427 7 4439 90838 54862 98804 155408 93994 306658 8 6069 5803676063 70594 101059 77539 208453 9 6463 70100 53821 102230 193410 146510419880 10 172388 176910 11 158959

[0044] TABLE D Parameters affecting signal strength as a function ofaperture rel. benzene input power sample area collection rel. depth Ksignal (992 rel. β Objective N.A. (mW) (cm{circumflex over ( )}2) angle(sr) (f/#) cm−1) (benzene) 10× 0.25 20 1.13 e−04 0.188 1.93 1 1 20× 0.4012 2.83 e−05 0.503 1.14 0.362 1.50 40× 0.75 10 7.07 e−06 1.759 0.440.038 0.57

[0045] TABLE E Relative changes in cross section (β) from resorcinarenethin film and nanoparticle arrays resorcinarene thin film 16 nm array 34nm array 42 nm array 70 nm array 87 nm array 111 nm array 170 nm arrayObjective (651 cm−1) (813 cm−1) (813 cm−1) (813 cm−1) (813 cm−1) (813cm−1) (813 cm−1) (813 cm−1) 10× 1 1 1 1 1 1 1 1 20× 1.52 3.04 2.65 3.873.29 3.52 2.75 2.91 40× <0.57> 4.90 7.43 4.48 3.91 5.44 4.01 4.64

[0046] Surface-Enhanced Raman Scatttering (SERS) Information

[0047] Surface-enhanced Raman scattering (SERS) integrates high levelsof sensitivity with spectroscopic precision and thus has tremendouspotential for chemical and biomolecular sensing. Efforts to understandand develop SERS as an analytical tool are dependent on methods forfabricating materials with stable and reproducibly high activities.Typical SERS substrates such as roughened gold and silver electrodes orcolloidal aggregates are disordered and often produce unpredictablesignal enhancements; moreover, their structures are unstable and quicklylose their SERS activity. Controlled methods for preparingnanostructured metal substrates may provide more useful correlationsbetween surface structure and signal enhancement. The particle size andinterparticle spacing are primary considerations for strong SERSactivity.

[0048] Considered here are the SERS properties of highly ordered,close-packed arrays of large (16-170 nm) gold nanoparticles. The SERSactivities from these substrates are tunable as a function of bothperiodic nanostructure and incident wavelength, with empirical signalenhancement factors (G) ranging from 10⁴ to over 10⁷. The goldnanoparticles in the mid-nanometer size regime (e.g. 15-200 nm) arecapable of self-organizing into planar close-packed arrays when treatedwith resorcinarene tetrathiol 2 as described above. The nanostructuredfilms vary in their optical reflectivities and absorptivities, changingin hue from blue to a faint grey. Extinction spectra revealsize-dependent shifts in dipolar plasmon resonance by hundreds ofnanometers from the visible to the near infrared (NIR) region (see FIG.2). The dramatic changes in extinction as a function of periodicitysuggest that the arrays are comparable to metallic thin films whoseplane surface plasmons have been localized by periodic gratings, andwhose optical properties also vary as a function of periodic structureand incident wavelength.

[0049] The SERS properties of the large gold nanoparticle arrays wereevaluated by comparing relative changes in the Raman signal intensitiesof adsorbed tetrathiol resorcinarene 2. Spectra were obtained from threeinstruments with different configurations and excitation wavelengths(λ_(ex)) but approximately equal solid angles of collection as definedby their effective numerical apertures (N.A.): a dispersive spectrometeroperating at 647.1 nm, a home-built Raman imaging microscope operatingat 785 nm (A. D. Gift et al., Raman Spectra, 30, 757-765 1999), and anFT spectrometer operating at 1064 nm (see FIGS. 3A, 3B). Empiricalsignal enhancement factors (G) were determined using peak integrationratios of the surface-enhanced Raman vibration of tetrathiolresorcinarene 1 at 813 cm⁻¹ to the corresponding unenhanced signal fromnoncrystalline films of defined thickness, with values ranging from 10⁴to over 10⁷ (see FIGS. 3C, 3D). It must be noted that the measurementsare macroscopic with respect to the periodicity of the arrays and do notnecessarily correlate with localized signal enhancements, which may beseveral orders of magnitude greater than the spatially averaged factors(see below). However, the G values reported here are practical fordefining minimum signal enhancements and can be used to evaluateperiodic trends.

[0050] Overall, unit particle size and excitation wavelength are foundto be strongly correlated with the SERS activity of the arrays (see FIG.3C). The extinction maxima of the arrays with the highest SERSactivities do not correlate strongly with λ_(ex), which suggestssignificant contributions by other means. These include scatteringefficiency, which is known to increase with particle diameter to thefourth power in isolated metal particles, and signal amplification byplasmon modes resonant at the Stokes-shifted Raman wavelength λ_(sr)which can differ from λ_(ex) by as much as 100 nm for the vibrationalband of interest.

[0051] Interestingly, the empirical enhancement factors demonstrate astrong dependency on the solid angle of collection. Micro-Raman spectraobtained at 785 nm, excitation using objective lenses with N.A.s of0.25, 0.40, and 0.75 yielded G values that varied by nearly an order ofmagnitude (see FIG. 3D). The effective Raman scattering cross section(β) of unenhanced samples did not change with aperture by more than afactor of 50%, confirming the surface-enhanced nature of the phenomenon.Values for β are often dependent on the angle of observation and alsothe angle of incidence, which have a strong influence on the electricfield intensity at metal surfaces. Raman scattering studies ofadsorbates on smooth and electrochemically silver surfaces havedemonstrated marked changes in signal intensities as a function ofobservation angle, with a maximum between 55° and 60° relative to thesurface normal. In comparison, the maximum half-angles from theobjective lenses above are 15°, 24°, and 49° respectively, whichcorrelate well with the considerable increases in G.

[0052] The resorcinarene-coated nanoparticle arrays are able to detectvolatile organic compounds with spectroscopic precision and thus havepromise as chemical sensors. Passage of approximately 30 mL ofnitrobenzene-saturated air through a flow cell containing the 170-nmparticle arrays resulted in the appearance of new peaks by SERS (seeFIG. 7). Signal intensities in the Raman difference spectra increasedwith further vapor exposure and are clearly associated with thevibrational modes of nitrobenzene (see FIG. 7). The analyte presumablyadsorbs to the resorcinarene surfactant layer in a nonspecific manner,so that its signal is enhanced solely by the electromagnetic field ofthe underlying nanostructured substrate. Other surfactant-coated SERSsubstrates have also demonstrated detection of organic analytes ingaseous and aqueous environments, with heightened sensitivity and/orselectivity in some cases.

[0053] Theoretical studies on aggregate structures suggest that theelectromagnetic field effects responsible for SERS are localized in theinterstitial regions of the arrays, accompanied by field depletionoutside of these areas. Recent SERS analyses of 50-60 nm metal colloidaggregates allege that NIR excitation produces giant local field effectscorresponding to Raman enhancement factors on the order of 10¹¹ or more.This implies that the great majority of the SERS signal from the arraysis due to the excitation of a very small percentage of adsorbate atthese interstitial sites, and that their true enhancements are greaterthan the surface-averaged G values by several orders of magnitude.Theoretical models of electromagnetic field intensities in periodicmetal gratings offer additional insights: the field enhancements withinthe channels are predicted to increase commensurately with aspect ratio,and to resonate at wavelengths determined by the grating amplitude. Suchmodels are congruent with the size-dependent SERS activities of thenanoparticle arrays: the interparticle spacings resemble gratingchannels with amplitudes and aspect ratios that vary as a function ofperiodic structure.

[0054] Some practical aspects of the large gold nanoparticle arrayspursuant to the invention follow. The nanostructured films of theinvention are stable in air and water at ambient temperatures and showno appreciable loss in SERS activity over a period of several months.The adsorption of volatile analytes is reversible, such that thesubstrates can be used multiple times. In addition, the SERSenhancements are reproducible for different sites on a given substrateas well as between replicate samples, with relative standard deviationsof 14 to 35% (see FIG. 3D).

[0055] As described above, an embodiment of the invention enables large(15-200 nm) gold particles to self-organize at the air-water interfaceinto monoparticulate films that can subsequently be transferred ontohydrophilic substrates as 2D hexagonal close-packed (hcp) arrays. Thecalixarene surfactant effects stabilization of these nanoparticleensembles; i.e. the calixarene surfactant layer is required to be highlyrepulsive at close range but thin enough to maintain minimalinterparticle separations, a critical parameter in the electromagneticproperties of metal nanoparticle assemblies. Short-range repulsion canbe enhanced by creating a surfactant layer with (i) hydrophobic chainsof uniform length at intermediate packing densities, such that theirrelatively high conformational entropies increase the barrier againststeric compression, and (ii) an appreciable surface charge density forgenerating repulsive electrostatic double-layer forces. These featuresalso render the encapsulated nanoparticles amphiphilic and promoteself-organization at the air-water interface. Calix resorcinarenes areexcellent surfactants for steric stabilization.

[0056] The large gold nanoparticle arrays have several other importantqualities that complement their size-tunable optical properties. Thearrays are stable in air at ambient temperatures and generatereproducible Raman signal intensities over a period of several months.The fabrication and transfer methods are straightforward and applicableto a variety of surfaces for a diverse range of optical functions. Thearrays are nanoporous by nature and may be used to isolate and studyindividual proteins and other macromolecules. Such practical featuresenhance the value of the nanoparticle arrays as substrates forapplications involving SERS and other surface-enhanced opticalphenomena.

[0057] The invention is not limited to making highly orderedmonoparticulate planar arrays of gold particles and can be practicedusing other metal nanoparticles. For purposes of further illustrationand not limitation, self-assembled rings of weakly ferromagnetic Conanoparticles (27 nm diameter) were treated with tetrathiolresorcinarene 4 and dispersed in toulene. The resorcinarene encapsulatednanoparticles self-assembled into 2D ring-like bracelets, and could bedeposited onto carbon-coated TEM grids (see FIG. 8). The Co nanoparticlerings may exhibit useful magnetic properties (e.g. stable chiralmagnetic domains) as a result of the collective behavior of magneticdipoles in cyclic configurations.

[0058] Another embodiment of the invention envisions using theresorcinarene-encapsulated planar arrays described above as substratesfor cell adhesion. It has been determined that several cell lines (BEK293, HeLa, and pancreatic β-cells) adhere strongly and remain viable onthe order of days on the gold nanoparticle arrays described above. Thenanoparticle arrays can be used to detect exogenous analytes asillustrated in FIG. 9, albeit at relatively high concentrations. Themethods described above can be used to fabricate gold nanoparticlearrays with greater interparticle spacings, which should delocalizefield effects and produce larger sampling volumes for lower detectionlimits.

[0059] Still another embodiment of the invention envisions cross-linkingthe resorcinarene surfactant layer on the encapsulated nanoparticles. Asillustrated in FIG. 10, tetraalkene resorcinarene 5, whose tails can becross-linked by olefin metathesis, can be used as the encapsulatinglayer on the nanoparticles. Gold nanoparticles encaged in cross-linkedshells were found to be resistant to precipitation-induced fusion, andcould even survive passage through a polystyrene size exclusion column.In lieu of tetraalkene resorcinarene 5, tetrathiol resorcinarene 6 ofFIG. 10 can be used in which cross-linking and polyvalent chemisorptionoperate synergistically to provide a robust, nondesorptive surfactantshell. Gold nanoparticles (20 nm) encapsulated by resorcinarene 6 werefound to be highly resistant to surface desorption when treated witholefin metathesis catalyst at low concentrations, while retainingexcellent dispersibility in organic solvents. These can be precipitatedand redispersed repeatedly in nonpolar solvents such as toulene andchloroform, and can be functionalized with organic or biologicallyactive ligands for site-directed nanoparticle delivery.

SYNTHESIS PROCEDURES

[0060] Synthesis of Tetrabenzylthiol Resorcinarene 1

[0061] Tetra-2, 2′, 2″, 2′″-methylresorcinarene. 2-methylresorcinol(0.687 g, 5.53 mmol) and dodecanal (1.2 mL, 5.44 mmol) were dissolved inEtOH (4 mL) and cooled to 0° C. in an ice water bath. HCl (4 N, 4.7 mL)was added dropwise and the reaction mixture was stirred for 10 minutes,then heated to 80° C. Within 10 min a yellow precipitate was observed.The reaction mixture was stirred for 4 days, then cooled to roomtemperature and poured into 40 mL of H₂O. The resulting precipitate wasfiltered, washed with water, then partially dispersed in MeOH andprecipitated to yield tetramethylresorcinarene as a yellow solid (0.863g, 55% yield). IR (neat, cm⁻¹): 3362 (br), 2916, 2849, 1474, 1458, 1335,1095. ¹H-NMR (DMSO-d₆, 300 MHz): δ 8.65 (s, 8 H), 7.18 (s, 4 H), 4.20(t, J=7.2 Hz, 4 H), 2.16 (m, 8 H), 1.94 (s, 12 H), 1.24 (m), 0.86 (t,J=6.9 Hz, 12 H).

[0062] Tetramethylcavitand resorcinarene. Tetramethylresorcinarene(0.482 g, 0.415 mmol) was dissolved in anhydrous DMF (21 mL) in aheavy-walled pressure reaction vessel. Bromochloromethane (1.1 mL, 16.9mmol) and Cs₂CO₃ (3.116 g, 9.56 mmol) were added and the reactionmixture was heated at 90° C. for 7.5 h. The reaction mixture was cooledto room temperature and poured into HCl (100 mL, 2 N) and extracted withdichloromethane (3×70 mL). The organic layer was washed with HCl (2 N,2×75 mL), water (100 mL), then dried over magnesium sulfate andconcentrated to dryness. Silica gel chromatography(hexanes/dichloromethane) yielded the desired cavitand resorcinarene asa white solid (0.352 g, 70% yield). IR (neat, cm⁻¹): 2926, 2853, 1468,1430, 1398, 1304, 1234, 1151, 1091, 1022, 980, 760. ¹H-NMR (CDCl₃, 300MHz): δ 7.00 (s, 4 H), 5.93 (d, J=6.9 Hz, 4 H), 4.77 (t, J=7.8 Hz, 4 H),4.27 (d, J=6.9 Hz, 4 H), 2.22 (m, 8 H), 1.99 (s, 9 H), 1.28 (m), 0.90(t, J=6.3 Hz, 12 H). ¹³C-NMR (CDCl₃, 75 MHz): δ 153.47, 138.17, 123.80,117.82, 98.72, 37.20, 32.18, 30.36, 30.10, 29.95, 29.63, 28.21, 22.93,14.33, 10.54.

[0063] Tetra-2, 2′, 2″, 2′″-(bromomethyl)cavitand resorcinarene.Tetramethylcavitand (0.094 g, 0.101 mmol) was dissolved in CCl₄ (3 mL)and treated with N-bromosuccinimide (0.101 g, 0.5674 mmol) andazo-bisisobutyronitrile (0.014 g, 0.085 mmol). The reaction mixture washeated to reflux at 85° C. for 5.5 h under an argon atmosphere. Thereaction mixture was then cooled to room temperature, diluted withdichloromethane (40 mL), washed with water (2×40 mL), dried overmagnesium sulfate and concentrated to dryness. Silica gel chromatography(EtOAc/hexanes) yielded the desired tetra(bromomethyl) resorcinarene asa white solid (0.088 g, 74% yield). IR (neat, cm⁻¹): 2925, 2853, 1471,1454, 1242, 1148, 1014, 974, 941, 668. ¹H-NMR (CDCl₃, 300 MHz): δ 7.14(s, 4 H), 6.03 (d, J=6.3 Hz, 4 H), 4.79 (t, J=7.8 Hz, 4 H), 4.57 (d,J=6.9 Hz, 4 H), 4.43 (s, 8 H), 2.21 (m, 8 H), 1.28 (m), 0.89 (t, J=6 Hz,12 H). ¹³C-NMR (CDCl₃, 75 MHz): δ 153.78, 138.28, 124.81, 121.27, 99.37,37.02, 32.14, 29.91, 29.60, 28.07, 22.90, 14.32. PDMS: expected forC₈₀H₁₁₆O₈Br₄=1525.4, observed=1525.7.

[0064] Tetrabenzylthiol resorcinarene 1. Tetra(bromomethyl)resorcinarene(0.258 g, 0.169 mmol) was dissolved in degassed DMF (11 mL) and treatedwith thiourea (0.082 g, 1.08 mmol). The reaction mixture was heated to80° C. under Ar atmosphere and stirred for 12 hours. The reactionmixture was cooled to room temperature and poured into degassed NaOHsolution (1 M, 40 mL), resulting in a white precipitate. The reactionmixture was stirred for 1 hour, then cooled to 0° C. and acidified to pH2 and extracted with EtOAc (3×50 mL). The organic layer was washed withH₂O (2×75 mL), dried over magnesium sulfate and concentrated to dryness.Silica gel chromatography (EtOAc/hexanes) yielded the desiredtetrabenzylthiol resorcinarene 1 as a white solid (0.152 g, 67% yield).IR (neat, cm⁻¹): 2924, 2852, 1469, 1016, 982. ¹H-NMR (CDCl₃, 300 MHz): δ7.07 (s, 4 H), 5.97 (d, J=6.9 Hz, 4 H), 4.76 (t, J=7.8 Hz, 4 H), 4.48(d, J=7.2 Hz, 4 H), 3.59 (d, J=7.5 Hz, 8 H), 2.2 (m, 8 H), 1.90 (t,J=7.5 Hz, 4 H), 1.28 (m), 0.90 (t, J=6 Hz, 12 H). ¹³C-NMR (CDCl₃, 125MHz): δ 153.12, 138.36, 127.40, 119.54, 100.13, 37.22, 32.16, 30.45,30.05, 29.92, 29.60, 28.14, 22.90, 18.32, 14.29. PDMS: expected forC₈₀H₁₂₀O₈S₄=1338.1, observed=1338.7.

[0065] Synthesis of Tetraarylthiol Resorcinarene 2

[0066] Tetraarylthiol resorcinarene 2. Tetrabromocavitand resorcinarenewas prepared by a modification of literature procedures.¹Tetrabromocavitand resorcinarene (0.576 g, 0.394 mmol) was azeotropedseveral times with toluene in a 2-neck round-bottomed flask. Thereaction flask was connected with a sidearm containing freshly sublimedsulfur (0.202 g, 6.304 mmol), then dried under P₂O₅ in vacuo overnight.The reaction flask was released to argon and treated with anhydrous THF(15 mL), then cooled to −78° C. and treated with n-BuLi (1.83 mL, 2.16 Min hexanes). The reaction mixture was stirred for 10 min at −78° C.,then treated with dry sulfur and slowly warmed to room temperature overa 6-hour period. The reaction mixture was treated with saturated aqueousNa₂S₂O₃ solution (15 mL), then diluted with water (150 mL) and extractedwith EtOAc (3×50 mL). The combined organic extracts were dried overmagnesium sulfated and concentrated to dryness. Silica gelchromatography (EtOAc/hexane) yielded the desired tetraarylthiolresorcinarene 2 as a white solid (0.432 g, 85% yield). IR (neat, cm⁻¹):2925, 2854, 1466, 1417, 1092. ¹H NMR (300 MHz, CDCl₃): δ 6.86 (s, 4 H),5.96 (d, 4 H, J=6.9 Hz), 4.74 (t, 4H, J=8.1 Hz), 4.37 (d, 4 H, J=7.2Hz), 3.78 (s, 4 H), 2.2-2.0 (m, 8 H), 1.5-1.2 (m, 72 H), 0.90 (t, 12 H,J=6.5 Hz). ¹³C NMR (CDCl₃, 75 MHz): δ 150.40, 138.46, 121.91, 98.53,37.47, 32.21, 29.97, 29.66, 28.07, 22.96, 14.38. Anal. Calcd forC₇₂H₁₁₂S₄: C, 71.21; H, 8.81; S, 10.00. Found C, 71.42; H, 8.75; S10.02.

[0067] Although the invention has been described with respect to certainembodiments, those skilled in the art will appreciate that changes,modifications can be made thereto without departing from the scope ofthe invention as set forth in the appended claims.

We claim
 1. A method for dispersing nanoparticles in a liquid medium bytreating said nanoparticles with calixarene as a surfactant.
 2. Themethod of claim 1 wherein said calixarene comprises a resorcinarene. 3.The method of claim 1 wherein said nanoparticles are encapsulated in anondesorptive shell comprising a crosslinked calixarene.
 4. A method formaking a self-assembly of nanoparticles by dispersing said nanoparticlesin a liquid medium using the method of claim
 1. 5. A method for making aself-assembly of nanoparticles by dispersing said nanoparticles in aliquid medium using the method of claim 1, forming a self-assembly ofthe treated nanoparticles, and then transferring said self-assembly ontoa substrate.
 6. A method for making a two-dimensional array of particleson a substrate, comprising treating the particles with a calixarene anddepositing the treated particles on a substrate as a two dimensionalarray.
 7. The method of claim 6 wherein the particles are treated byreaction with calixarene molecules.
 8. The method of claim 6 wherein thecalixarene comprises a resorcinarene have a large, concave headgroup. 9.The method of claim 8 wherein the resorcinarene comprises tetrathiolresorcinarene.
 10. The method of claim 6 wherein the particles aremetallic.
 11. The method of claim 6 wherein the particles have adiameter of 15 to 200 nm.
 12. A two-dimensional particle assembly on asubstrate wherein said assembly comprises calixarene-treated particles.13. The array of claim 12 wherein the particles compriseresorcinarene-treated particles.
 14. The array of claim 12 wherein theparticles are metallic and have a diameter of 15 to 200 nm.
 15. Achemical or biomolecular sensor including the assembly of claim 12.